Prions produce brain degeneration in people and many species of farmed and wild animals. Despite considerable progress in understanding the biology of prions and the CMS diseases caused by these infectious proteins, much remains to be learned. Investigations of mammalian prions continue to be slow since mouse bioassays generally require incubation times exceeding 100 days, and are very expensive due to the high levels of biosafety that are required. Two recent discoveries promise to accelerate many avenues of research; these are: (1) the length of the incubation time in mice is directly proportional to the stability of the prion strain and (2) neurospheres produced from the embryonic brains of transgenic (Tg) mice overexpressing PrP can support prion replication. In this application, we propose to identify conditions that make neurospheres susceptible to infection with prions. Neurospheres will be derived from Tg mice expressing full-length and truncated PrPs. We plan to investigate the rate of prion propagation in neurospheres using three well defined isolates covering a range of conformational stabilities. After neurospheres are exposed to prions, they will be cultured for an appropriate interval before the PrPSc levels in the cells are determined by immunoassay. We anticipate that these studies will be complex since the relationship between the length of the time interval from inoculation to PrPSc measurement is likely to vary inversely with the number of prions in the inoculum and the rate of prion replication is predicted to be inversely proportionalto the stability of the strain. We plan to manipulate the culture conditions and modify the prion inocula to understand the factors influencing neurosphere susceptibility to prions. From these studies, we hope to determine the relationship between the rate of prion propagation in neurospheres and the stability of the prion strain. Neurosphere cultures will then be produced from Tg22372, Tg440, Tg4092, Tg14882 and Tg12584 mice expressing chimeric Hu/MoPrP, wt HuPrP, BoPrP, OvPrP and ElkPrP, respectively. Once the neurosphere cultures are established, they will be infected with naturally occurring CJD, BSE, scrapieand CWD prions. We propose to optimize conditions for the replication in the neurospheres of naturally occurring human, bovine, ovine and cervid prions and use the neurospheres to develop sensitive, rapid, low cost bioassays for these naturally occurring prions.